Purification of streptomycin



United States Patent 2,767,168 PURIFICATION OF STREPTOMYCIN Lee C. Cheney, Fayetteville, N. Y., assignor to Bristol Laboratories Inc., Syracuse, N. Y., a corporation of New York No Drawing. Application February 20, 1953, Serial N0. 333,129

4 Claims. (Cl. 260-210) This invention relates to a new class of organic chemicals useful in therapeutics and in the manufacture of streptomycins and, more particularly, to new 1,3-substituted-2-streptomycyl-tetrahydroimidazoles and their nontoxic salts.

Throughout this specification and claims, the names streptomycyl and hydroxystreptomycyl are used to represent the radicals attached to the aldehyde group in the widely-known antibiotics streptomycin and hydroxystreptornycin. Thus, the antibiotic streptomycin is represented by the formula Streptomycyl-CHO or as Strep-CHO (see U, S. Patent #2,607,770) rather than by the customary formula. An ordinary salt, such as the sulfate, is represented as Streptomycyl-CHO- 1 /2H2SO4 or as 2 Streptomycyl-CHO-3H2SO4.

Up to the present time, there have not been available any active, non-toxic salts or forms or derivatives of streptomycin which are relatively insoluble in water. In chemical production, this means that elaborate processes, such as those using ion-exchange columns, are required to concentrate a solution of crude streptomycin. Final crystallization is a complex and expensive process using large amounts of organic solvents and generally also requiring formation of a calcium-chloride double salt before a satisfactory crystalline sulfate can be prepared. Illustrative processes are given by U. S. Patents #2,446,l02, 2,541,420, and 2,578,840. Expensive toxic dyes are used by Regna; see U. S. Patents #2,555,762, 2,555,763, 2,560,889, 2,560,890, 2,555,761, 2,555,760.

A further disadvantage of known salts and derivatives of streptomycin and the free base is their instability under alkaline conditions, which irreversibly decompose and inactivate the streptomycin, forming the gamma-pyrone, maltol.

In therapeutic use, present water-soluble salts and derivatives of streptomycin and the free base are rapidly absorbed when administered by injection, e. g. intramuscularly, and do not give protracted blood levels. They are also absorbed very poorly upon oral administration. In the treatment of tuberculosis, prolonged high concentrations of streptomycin are not necessary and in addition increase the possibilities of neurotoxic reactions (Antibiotic Therapy, Welch and Lewis, Arundel Press, Washington, D. C., 1951, page 104) A repository preparation giving prolonged but not too high blood levels is therefore desired to overcome these objectives and to spare the patient and physician the inconvenience and expense of numerous injections.

It is an object of the present invention to provide a new series of derivatives of streptomycin and hydroxystreptomycin and their acid addition salts, which are nontoxic, therapeutically eiiective, relatively insoluble in water, stable in aqueous alkali and easily regenerated by aqueous acid to the original soluble, active streptomycin.

It is a further object of the present invention to provide improved processes for the isolation and purification, as from inorganic and ash-forming impurities, of streptomycin from crude, aqueous solutions.

It is a further object of the present invention to provide a new series of derivatives of streptomycin and hydroxystreptoinycin and their non-toxic acid addition salts which upon parenteral administration in suitable media provide prolonged blood levels and prolonged therapeutic action.

It is an additional object of the present invention to provide a new series of derivatives of streptomycin and hydroXy-streptornycin and their non-toxic acid addition salts which are systematically absorbed and effective upon oral or buccal, e. g. sublingual, administration.

The objects of this invention have been achieved and there is now discovered, according to the present invention, the new series of compounds selected from the group consisting of compounds having the formula 3 R4 tat-91H lit-I l 1 1'R2 0 s treptomycyl wherein R1 and R2 represent radicals selected from the group consisting of alkyl, cyclopentyl, cyclopentyl-lower alkyl, cyclohexyl, cyclohexyl-lowe'r alkyl, lower alkylcyclohexyl-lower alkyl, lower akoxy-cycohexyl-lower alkyl, lower alkyl-cyclohexyl, lower alkoxy-cyclohexyl, dehydroabietyl, pyridyl, lower alkyl-pyridyl, thiophenelower alkyl, lower alkyl-thiophene-lower alkyl, furanlower alkyl, lower alkyl-furan-lower alkyl, quinolyl-lower alkyl, naphthyl, benzhydryl, piperonyl, thiazolyl, phenyl, lower alkyl-phenyl-lower alkyl, phenyl-lower alkyl, halophenyl-lower alkyl, nitrophenyl-lower alkyl, hydroxyphenyl-lower alkyl, I-IzN-phenyl-lower alkyl, lower alkoxyphenyl-lower alkyl, alkoxy-hydroXy-phenyl-lower alkyl, and di-lower alkyl-monohydroxy-phenyl-lower alkyl; R3 and R4 are members selected from the group consisting of hydrogen and methyl; and (b) acid addition salts thereof.

The products of the present invention are useful, nontoxic therapeutic agents in diseases of man and animals caused by streptomycin-sensitive tuberculosis bacteria and gram-negative bacteria and are useful as growth-stimulating supplements for animals and poultry, as in feeds, and for use to stimulate the growth of plants, such as radishes, oats and grass.

Further, as illustrated in detail below, the products of the present invention are of particular value in the commercial production of streptomycin because, by virtue of their very low solubility in water and other solvents, they provide improved means for isolation and purification of streptomycin from impurities, e. g. ash-forming impurities.

The products of the present invention are further use- 111 for their ability to provide prolonged, therapeutic blood-levels upon parenteral injection in comparison with the non-toxic streptomycin salts of the prior art.

The products of the present invention are prepared by the reaction of hydroxystreptornycin free base, streptomycin free base or water-soluble acid addition salts thereof, and I prefer streptomycin sulfate, with an equimolar quantity, or more, of an N,N-substituted-m,-diamino alkane, where there are two to four carbon atoms in the alkane carbon chain. Two examples are N,N-diben zylethylenediamine and N-p-methylbenzyl-N'-benzyl.-l,2-di+ aminopropane. I prefer to conduct the reaction in water but can also use a mixture of water and a water-miscible organic solvent such as methanol or acetone when desired, e. g. to solubilize' the amine, The reaction takes place at room temperature over a period varying from several hours to several days but can be accelerated by heating, e. g. to 50 C. for twenty minutes. The mixture needs to be maintained at a pH of about 7.0 or higher, since acid conditions reverse the reaction andregenerate the starting reagents. The product precipitates as a'solid or as an oil which solidifies on seeding. vThe product may be purified of unreacted streptomycin reagent, i. e. sulfate, by slurrying with water.

Briefheating in aqueous acid regenerates the starting materials and often precipitates the diamine salt, e. g. N,N-dibenzylethylenediamine hydrochloride. The purified streptomycin can be recovered in the usual ways, as by lyophilization or by crystallization of the sulfate by the addition of alcohol.

This invention also includes all acid addition salts for processing purposes and all non-toxic acid addition salts for therapeutic purposes, organic and inorganic examples of which include hydrochloric, sulfuric, sulfamic, tartaric, hydrobromic, hydriodic, glycolic, citric, maleic, phosphoric, succinic, acetic, benzoic, cinnamic, mandelic, malic, ascorbic, and the like. I prefer the sulfate.

When the irnidazoles of the present invention are used for therapeutic purposes, they may be used in water with a suspending agent such as sodium carboxymethylcellulose, Spans, Tweens, lecithin orpectin or in injectable oils, e. g. peanut oil gelled'with aluminum monostearate in a manner analogous. to the art pertaining to insoluble salts of penicillin. If desired, suspending or dispensing agentsmay be added to increase pharmaceutical elegance. As a suspending or dispersing agent, sodium carboxymethylcellulose has been found highly satisfactory but carboxymethylcellulose, methylcellulose, polyvinylralcohol, polyvinylpyrrolidone, gum tragacanth, gelatin, pectin, sodium alginates, dextran, gum Karaya, the

copolymer of methyl vinyl ether and maleic anhydride, and'the like, are also useful. The amount of suspending agent will vary to a certainextent, but usually from about O.2 to.5.0 percent, preferably from 0.5 to 2.5 percent, is'employed and variations within these ranges maybe made by any experienced chemist or pharmacist with regard to the intended use of the composition. Thus the. concentration of polyvinylpyrrolidone may vary from 0.1% to 25%, with about 10% preferred. The concentration of dextran may vary from 0.1% to 20%, with about 10% preferred. The concentration of pectin may vary from 0.1% to' 0.5%, with about 0.2% preferred. The concentration of gum tragacanth may vary'from 0.5% to 2% with about 1% preferred; 5% sodium chloride may be added theret It is to be understood that the words agent and dispersing agent to describe the additives such cellulose, lecithin, Spans and Tweens which improve the pharmaceutical elegance of these preparations, as by increasing ease of injection and ease of resuspension upon settling. Other suspending and dispersing agents include lecithin, Falba, cholesterol, Span 20, Span 40,, Span 60, Span 80, the Tweens, Amerchols, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, alginic acid, propylene glycol alginate, polyoxyalkylene derivatives of sorbitol fatty acid esters, urea and sodium p-aminobenzote. V V

The composition is not limited to the exact ingredients previously described and to the exclusion of all others, since various other ingredients, 'While not necessary, may be added, if desired. For instance, asmall amount of preservative, such as Phenol U. S. P., Cresol U. S. P., Methyl Paraben (methyl ester of p-hydroxybenzoic acid), Ethyl Paraben (ethyl ester of p-hydroxybenzoic acid),

suspending are used interchangeably as sodium carboxymethyl- Butyl Paraben (butyl ester. of p-hydroxybenzoic acid) or Propyl Paraben (propyl ester of may beemployed. Other ingredients which improve added. Examples of such ingredients are lecithin, Falba, cholesterol, Span 20, Span 40, Span 60, Span 80, Tween 20, Tween 40, Tween 60, Tween 80, Tween 85, Amerchols, urea, and sodium para-amino-bnzoate. The Spans are hexitol anhydride (hexitans and hexides derived from sorbitol (partial'esters of common long-chain fatty acids (e.

'g.,*la'uric, palmitic,.stearic, :oleic) and the Tweens are polyoxyalkylene derivatives of' the Spans. V

The diamine reagents'of this invention are prepared by the usual synthetic-methods. .-Thus by the reaction of ethylene dichloride with an appropriately substituted amine, substituted ethylene diamines are formed. Alternatively, ethylenediamine or its acid addition salts are reacted with formaldehyde and a suitably substituted compound containing an active hydrogen atom, such as aldehydes, ketones, thiophene, organic acids and the like, whereby N,N-disubstituted ethylene diamines are obtained. In another method for thepreparation of substituted ethylene diamines, a suitably substituted aldehyde or ketone 'is condensed with ethylene diamine 'to form .the correspondingly substituted diimine' The diimine is hydrogenated by the usual methods, such as catalytic reduction and the like, to give the desired substituted ethylene diamine. Acid addition salts of the substituted ethylene diamines can be prepared by the methods of the art, as, for example, by inter-reaction of equivalent amounts of the substituted diamine base and a selected acid in inert solventsolution, followed by re moval of the solvent;

Thus 1,2-bis-(phenylethylamino)ethane is prepared by mixing a solution of 54 g. (0.5 mole) of 1,2-dibromoethane in 250 cc. of ethanol and 242 g. (2 moles) of 2- phenyl-ethylamine. The mixture is boiled for about an hour, made alkaline with potassium hydroxide and boiled for a further lO-minuteperiod. The precipitated potas sium bromide is removed the alcohol is evaporated off and the residue, comprising 1,2-bis-(phenylethylamin'o)- ethane, is fractionally distilled under pressure. 1,2-bis- (phenylethylamino)ethane boils at about 240 C. at the pressure of 15 of mercury.

Thus, in a 500 ml. three-necked flask, fitted with stirrer, condenser and thermometer, are mixed 42 g. (0.5 mole) of thiophene, 33 g. (0.25 mole) ethylenediamine dihydro.

chloride and 43 ml. of 36 aqueous formaldehyde The mixture is stirred and heated to gradually raise the temperature. At 60 C. a vigorous reaction begins, heating is stopped and an ice-bath is applied to the flask. The internal temperature rises to about 73 C. and the reaction mixture solidifies. 200 m1..of 50% aqueous alcoholis added and stirred and the mixture is heated an additional minutes. After cooling, the reaction product is filtered and washed with water. amorphous and dissolved in 250 ml. hot water, cooled and made alkaline with 40% sodium hydroxide. The free base which separates is not very soluble in ether and is taken up in benzene, dried over sodium hydroxide and is obtained as a colorless, viscous oil on removing'benzene in vacuo. The oil is converted to diacetate by dissolving in 200 ml. ethyl acetate and adding 12 glacial acetic acid. The precipitated N,N-bis-(2-thenyl)-ethylenediamine diacetate is collected by filtration, Washed with ethyl acetate and found to melt at about 84C.

In essentially the same manner as above, one may also react an alkylene diamine such as ethylenedia'mine and an acid, as, for example, hydrochloric, sulfuric or formic acid, to form the di-acid salt together with half a' mol. of 7 p-hydroxybenzoic acid) The white product is a Mixed N,N'-monos"ubstinited-ethyiene diamines may be prepared in the ways known to the art. Thus, to prepare N-benzyl-N-alpha-ethyibenzyl-ethylenediamine, dimethyl benzyiaminoacetal (49 g 0.25 mol.) and I-phenylprfopyla'mine (34 g., 0.25 mol.) are mixed in a 500 ml. flask fitted with a reflux condenser with a drying tube. Forrnic acid (75 ml, 98100%) is added all at once. A vigorous reaction ensues with darkening and evolution of heat and of carbon dioxide. When the initial vigorous reaction has subsided, the mixture is heated to reflux for two hours and excess formic acid isthen removed by distillation under reduced pressure to leave a tarry residue to' which is added 150 ml. of 6 N HCl. After heating for reflux for one hour, cooling in an ice-bath and making alkaline by the addition of 40% aqueous sodium hydroxide', the supernatant layer is separated, diluted with 400 ml; ether and filtered to remove tar. The filtrate is dried over sodium hydroxide and treated with methanolic hydrochloric acid to precipitate N-benzyl-N-alpha-ethylberi'zyl'ethylenediamine dihydrochlo'ride, which melts at about 305 C. after recrystallization from aqueous methsnot.

Numerous other N,N'-bis-(mono-substituted)ethylenediamines may be prepared according to U. S. Patent #2,627,491. Formation of N-cyclohexyl-N-ethyl-ethylenediarnine is taught by this patent by reaction of acetaldehyde with N-cyclohexyl-ethylenediamine followed by catalytic hydrogenation; N-benzyl-N'-vanillylethylenediamine is prepared by reaction of N-benzylethylenediamine with vanillin followed by reduction with formic acid. In the same patent are disclosed numerous N-monosubstituted-ethylenecliamines (e. g. N-heptylethylene'diamine) and a general process for their preparation by reaction of an aldehyde (e. g. heptaldehyde) with ethylenediam'ine diforrnate. These products can in turn be reacted as their diformates with aldehydes to give diamines 6 EXAMPLE I 1,3-dibenzyl-2-stieptomycylteti'ahydfoimidazolesulfate To a solution of 7.3 g. (0.01 mole) of streptomycin sulfate in 50 ml. of Water was added 5.4 g. (0.0225 mol) of N,N-dibenzylethylenediamine in 25 of methanol. Addition of ml. of methanol gave a clear solution. Heating on the steam bath at 45-50 C. for ten minutes caused precipitation of l,3-dibenzyl-Z-streptomycyltetrahydroimidazole sulfate as an oil which crystallized rapidly to a white solid. It was chilled, filtered and air-dried; weight-8.4 g.

The product was slurried for a few minutes with ml. of water to insure removal of all streptomycin sulfate, collected by filtration and air-dried. Weight--7.2 g.; M. P.-243 247 C. (decomposition, with previous browning); solubility-A980 u./ ml. or about 8 mg'r'nsjml; potency-5l2 u./rng. (87% of theory).

Analysis.Calculated for C37H5'1N9O11- 1 /2 H2804:

Calculated Found A 1.5 g. sample of the imidazole was suspended in 25 ml. of 6 N HCl and heated for three hours at 100 C. The mixture was filtered and the solid product recrystallized from water to give 0.5 g. of N,N'-dibenzylethylenediamine dihydrochioride. M. P.305306 C. (d.). An authentic sample gave M. P. 305 307 C. ((1.). The filtrate contained active, regenerated streptomycin in solution.

EXAMPLE II The procedure of Example I was repeated with certain variations and the following, tabulated results.

Grams of Amine- Potency '(biE-i Solubility in water of Reaction Time 3.6 245255 C. ((1.) 630 u./mgm

omit m1.

. 'iibiri'iri' for 45-50 0. mi: 10 few hours. days. minutes; Room temp. 24 hours.

1 Product slurried with 50 ml. water to remove unreacted streptomycin sulfate, collected by filtration, air'dried and then weighed and assayed. v

2 The potency of the product was determined on suspensions by bio-assay (subtilis and coli) due to its insolubility in water, methanol, butanol, ketones, acetonitrile, nitromethane, chloroform, dimethyl iormamide and amyl acetate and acetone. The suspensions contained 10 mgms. of finely-ground product per ml. water.

3 The solubility of the product was determined by shaking at least 0.1 g. of finely ground material in 10 ml. water for hours, filtering the solution which contains much undissolvedproduct and assaying the filtrate (subtilis and colt).

of the formula RiNH-CH2-CH2NHR2 where R1 and R2 difier.

This application is a continuation-impart of copending application Serial No. 327 ,45 6 of Buckwalter and Cheney, filed December 22, 1952, directed to the utilization of N,N-bis-(dehydroabietyl) ethylenediamine as the N,N- bis-(dehydroabietyl) ethylenediamine dipenicillin G. salt.

Further understanding of the invention may be obtained by reference to the following examples which are illustrative only and are not the exclusive embodiment of the invention. I Wish it to be understood that I do not desire to be limited to the exact details shown and described, for obvious modifications will occur to a person skilled in the art.

EXAMPLE III 1 ,3-di fi-phenethyl -2-strept0mycyltetrahydroimidazole sulfate M. P. 195 205 C. (d.; browns at C.); solubility 7 in water-4400 u./ml.; potency396 u./mg. (67.5% of theory). 7

' Analysis-Calculated for Cs9Ha1N9O11-1 /2H2SO4:-

Calculated Found on--. 47.9 48.1 H 6.6 6. 9

Y EXAMPLE IV 1,3-di(dehydr0abietyl) -2-strept0mycyltetrahydroimidazole sulfate A solution of 3.0 g. (0.005 mole) of N,N-di(.dehy-.

EXAMPLE V 1,3-dicyclohexyl-Z-strept0mycyltetrahydroimiclazole sulfate A solution of 2.4 grams (0.011 mole) of N,N'-dicyclohexyl-ethylene-diamine dissolved in the minimum amount of methanol is added to 7.3 g. (0.01 mole) streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand. at room temperature for three days. The product, 1,3-dicyclohexyl-2-streptomycyltetrahydroimidazole sulfate, precipitates either upon cooling or upon the addition of water and is collected by filtration or by decantation. 7

- A 15 g; sample of this imidazole is heated for three hours at 100 C. in 25 m1. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

. EXAMPLE VI 1,3-di-tert.-butyl-Z streptomycyltetrahydroimidazole sulfate A solution of 1.8 grams (about 0.01 mole) of N,N-ditert.-butylethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. (0.01 mole) streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3 di-tert.-butyl-2-streptomycyltetrahydroimidazole sulfate, precipitates either upon cooling or upon the addition of Water and is collected by filtration or by decantation.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE VII l,3-di-(,1,3,3-tetramethyl-n-butyl) -2-strept0mycyl- 'tezrahydroimidazole sulfate Recovery-5.0 g.; potency-185 u./mg.'

droimidazole-sulfate, precipitates either upon cooling or upon the addition of; water and is collected by filtration or by decantation.

A 1.5 g. sample of this imi dazole is heated for three hours at C. in 25 ml. of 6 N HCl to. regenerate the amine hydrochloride and soluble, active streptoniy cm.

EXAMPLE VHI J,3-di-n heptyl-2-streptomycyltelrahydromia'azole sulfate A solution of 2.7 grams (about 0.01 mole) of'N,N.-. di-n-heptylethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. (0.01 mole) streptomycin sulfate dissolved in 50 ml. water. The min imum amount of ethanol is added to ensure complete solution. After heating at 45 50, C. for ten minutes, the reaction mixture is allowed to stand at room tern perature for three days. The product, 1,3-di-n-heptyl-2- streptomycyltetrahydroimidazole sulfate, either upon cooling or upon the addition of water and is collected by filtration or by decantation.

A 1.5 g. sample of this imidazole is heated for three hours at 100 'C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE IX 1,3-di-benzhydryl-Z-sireptomycyltetrahydroimidazole sulfate A solution of 4.3 grams (0.01 mole) of N,N-di-benz-' hydrylethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. (0.01 mole) streptomycin sulfate dissolved in 50 ml. water. 'The minimum amount of ethanol is added to ensure complete solution. After heating, at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-di-benzhydryl-2-streptomycyltetrahydroimidazole sulfate, precipitates either upon cooling 'or upon the addition of water and is collected by filtration or by decantation.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin...

7 EXAMPLE X I,S-dipheny[-2-streptomycyltetrahydroimidazole sulfate A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycinQ 7 EXAMPLE N ,N '-bis-dehydroabietylethylenediamine Dehydroabietylamine grams), ethylene bromide (59.3 g.) and potassium carbonate (92 g.) were mixed with 2.5 liters toluene in aflask equipped witha stirrer" and refluxed overnight. The toluene solution was filtered,

washed with dilute sodium hydroxide and then two liters of Water and the toluene removed by distillation to leave 175.3 g. .crude product, N,N-bis-dehydroabietylethylene diamine which was purified by distilling off the impurities, particularly dehydroabietylamine, up to 275 C./l mm., at which point the product begins to distill.

precipitates arenas A mixture of 650 grams (5.0 moles) of t-octylamine (Rohm and Haas, B. P. 137-142" C. at.760 mrnr. also called l-amino-l,1,3,3-tetramethyl-n-butane) and 50 ml. of water was heated under reflux with Stirring during the dropwise addition of 188 grams (1.0 mole) of ethylene dibromide, and heating was continued for an additional 8 hours. While still warm, the mixture was treated with 300 grams of 50% sodium hydroxide; the organic layer was separated and washed with dilute alkali and water. After drying, the material was fractionally distilled to yield about 260 grams (about 92% of theoretical) of N,N'-bis(i-octyD-ethylenediamine boiling at 120 C. at a pressure of 0.5 mm. Hg.

EXAMPLE XIII N,N'-bis=(cyclahexylmthyljerhylendiamine Commercially available 1,2,5 ,fi-tetrahydrobenzaldehyde (30.0 grams; 0.272 mole) in 50 ml. methanol is mixed with 7.5 grams (0.125 mole) ofarihydroiis' e'thyletiediamine. Apprecia'ble heat is evolved and the solution turns yeltow. Raney niokel catalyst- (40.0 grams wet weight) washed successively with water and methanol is added and the mixture is hydrogenated with shaking in an atmosphere of hydrogen at about 50' pounds per square inch gauge at about 60 C. After the absorp tion of about two moles of. hydrogen for each mole oftetrahydrobenzaldehyde used, the catalystis removed by filtration and the solution is acidified with concentrated hydrochloric acid. White crystalline N,N-bis(c'yclohexylmethyl ethylenediamine dihydrochloride precipitatesand is collected. Upon recrystallization from water, this product melts at about 318 3 19 C. with decomposition.

Analysis. Calculated for C1eH32N2=2HCl-:

-Galct'ilated Found N,N'-bis-(cyclohexylniethyl)thylendiamine dihydrochloride (3.2 grams, 0.01 mole) prepared as suave. is suspended in Water and neutraliied 50% sodiiiiti hydroxide. The liberated fr'ee base, N,N'-bis-'(cyclohexyl-' methyl)ethylenediamine, is extracted into ether'. The combined ethereal extracts are washedwitli water and saturated sodium chloride solution filtered through anhydrous sodium sulfate. The solvent other is removed by' distillation, leaving the free base as a colorless oil.

EXAMPLE XIV dinin'ir'ie Anhydrous ethylenediamine (30.0 g.-, 0.5 mole) is added to a solution of 4-hydroxy-1,S-dimethylbenzene (122.2 g., 1.0 mole) in 250 ml methanol. Formalin U. S. P. (75 mL, 1.0 mole) is added dropwise while the mixture is stirred and refluxed for nineteen hours. After adding 200 m1. of concentrated hydrochloric acid; followed by cooling and the addition of toluene to facilitate handling, the white, crystalline dihydrochloride of N,N'- bis-(3,S-dimethyl-ZhydroxylienZyIj-ethylenediamiii firecipitates and is collected by filtration. v

After two recrystallizations' from; a mixture prepared from equal volumes of water and methanol to whicfli about one-twentieth part of concentrated hydra-emetic acid has been added, the product melts at about 225.5

228.5 c. g g V Analysis.Calculated for Ciel-istiCltNzQir 1 0 The free base is liberated from the dihydrochloride with alkali in the usual manner.

EXAMPLE N,N'-bis(alpha-methylbeiizyl -ethyleriediziiriifi Alpha methylbenzylamine (48.4 grams,- 0.4 mole) is placed in a three-necked flask fitted with stirrer, cof denser and dropping funnel and arranged for heating with an electric mantle. Ethylene dibromide (18.8 grams, 0.1 mole) is dropped into the heated amine over a period of two hours. After all of the bromide is added, 8 of water is added and the reaction mixture is refluxed for an additional six hours. The mixture is allowed to coolto' room temperature and is then neutralized by the addition of ten grams of sodium hydroxide in 25 ml-.- of water. The organic layer is separated, dried and distilled under reduced pressure to give N,N'-bis(alpha-methylbenzyl)ethylenediamine,- boiling about 176-185 C. at 4 mm. of mercury pressure.

EXAMPLE XVI 1.3-dipiperonyl-2 strptomycyl-tetmhydroimidazdle sulfate A solution of 3.3 grams of N,N'-dipiperonylethylenediamine (M. P. about 145 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sul fate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for threedays. Lyophilization of the reaction mixture leaves solid 1, -3 di- (piperonyl) -2-streptomycyl tetrahydroimidazole sulfate.

A 15 sample of this imidazole is heated for three hours at C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble active streptomycin.

EXAMPLE 1,3 di(furfuryl) 2 streptomycyl tetrahydroimidazole sillfdte A solution of 2.2 grams of N,N'-difurfurylethylenediamine (M. P. about /0.l5 mm.) dissolved in the minimum amount of methanol is added to 7,3 g. streptomyc'in' sulfate dissoly'edin 50 ml. water. The amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation invaded below 50 C. and replaced by water; upon cooling the product, 1,3'-'di(furfuryl)-2-streptomycyl tetrahyd'roimidazole sulfate, precipitates as a gum which gradually solidifies upon seeding.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XVIII 1 ,3-'bis-(2-h eptyl -2-'strep tbmycyl-tetrahydroimidazole" sulfate A- solution of 2.6 grams of N,N-bis (2'-heptyl)'-ethylene'diamine (13. 1. about 126 C./ 2 mm.) dissolved in the minimum aniouiitof methanolis added to 7.3 g. strpto ""f" 75 amine hydrochloride and soluble; active streptom'y'eih.

11 EXAMPLE/ XIX 1,3 bis(gamm,a phenylpropyl) 2 streptomycyl tetrahydroimidazole sulfate 7 A solution of 3.0 grams of N,N'-bis(gamma-phenylpropyl)-ethylene-diamine (B. P. about 160 C./0.25 mm.) dissolved in the. minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water.

cyl-tetrahydroimidazole sulfate, is filtered off anddried;

.gA 1.5'g. sample of this imidazole 'is heated for three hours at 100 C. in 25 m1. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XX 1,3-di(cycl0hexyl) -2-streptomycyl-tetrahydroim iddzo le sulfate" Afsolution of 2.2 grams of N,N'-dicyclohexylethylenediamine (M. P. of hydrateabout 82 C.; dihydrochloride melts about 315 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in SO ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-di-(cycloheXyl-Z-streptomycyl-tetrahydroimidazole sulfate, is recovered as a nearly white solid by removing the solvents by distillation in vacuo.

'A 1.5 g. sample of this imidazole. is heated for three hours at 100 C. in 25 ml..of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

L3-bi s- 4-methyl-2-pentyl) 2-streptjornycyl-tetrahydroimidazole sulfate A solution of 2.3 grams of N,N'-bis-(4-methyl-2- hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE xxn 1 ,3-bis-(3,5 ,5 -trimethylhexyl )-2-streptomycyl-tetrahydroimidazole sulfate A solution of 3.1 grams of N,N-bis-(3,5,5-trimethylhexyl)-ethylenediamine (B. P. about 150/ 1.2 mm.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction'mixture is allowed to stand at roomtemperature for three days. The product, 'l,3-bis-(3,5,5-' trimethylhexyl) 2-streptomycyl-tetrahydroimidazole sulfate, is recovered as a solid by'lyophilization.

A 1.5 g. sample of'this imidazole is heated for three hours at 100 C. in 25 ml. of-;6 N HCl'to regenerate the amine'hydro chloride and'soluble, active streptomycin.

. ture for three days.

aromas 1 .2 EXAMPLEXXIII 1,3-bis- (p-chlorobenzyl) -2-strep tomycyl-teirahydroirniulazole sulfate I A solution of 3.1 grams of N,N-bis-(p-chlorobenzyl)- ethylenediamine (diacetate melts about 126 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten min- 7 'utesQthe reaction mixture is allowed to stand at room temperature for three days.. The precipitate of l,3-bis-' (p-chlorobenzyl) .2 streptomycyl-tetrahydroimidazole sulfate which separates upon the addition of water is collected by filtration and dried. I 1 Y A 1.5 g. sample of this imidazole is heated for three hours at C. in 25 ml. of 6 N HCl to regenerate'the amine hydrochloride and soluble, active streptomycin;

EXAMPLE XXIV 1,3-liis- (2,4 dihlo'robenzyl) -2-str eptomycyl-tetrahydroimidazole sulfate 7 i A solution of 3.8 grams of N,N'-bis-(2,4-dichlorobenzyl)ethylenediamine (picrate melts about 183 C.) disa hours'at 100 C. in'25 ml. ,of6 N HCl to regeneratethe amine hydrochloride and soluble, active, streptomycin.

EXAMPLE XXV 1,3-bis- (p-rtitr0benzyl)-2 streptomycyl-tetrahyd ioimiduzole sulfate 'A solution of 3.3 grams of N,N-bis-(p-nitrobenz'y1)- ethylenediamine (dihydrochloride melts about 290 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water, The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation in vacuo below50 C. and replaced by water; upon cooling the product, 1,3-bis:(p-nitrobenzyl)-2- streptomycyl-tetrahydroimidazole sulfate, precipitates as a gu m which gradually solidifies upon seeding.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXVI 1 ,3-bis-'(p-uminobenzyl)-2-streptomycyl-tetrahydroirr idazole sulfate A solution of 2.7 g. N,N-bis-(p-aminobenzyl)-ethylenediamine (dihydrochloride melts above 300 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed V The product, .1,3-bis-(p-aminobenzyl)-2-streptomycyl-tetrahydroimidazole sulfate, precipitates upon the addition of Water and is filtered off and dried. a

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N amine hydrochloride and'soluble, active streptomycin.

to stand at room tempera- HCl to regenerate the 13 EXAMPLE XXvu 1,3-bis-(p-methoxybenzyl} -2-strep tomycyl-tetrahydroimidazole sulfate A solution of 3.0 grams of N,N-bis-(p-methoxybenzyl) -ethylenediamine (dihydrochloride melts about 285 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. forten minutes, the'reaction mixture is allowed to stand at room temperature for three days. Upon cooling and the careful addition of water, the solid 1,3-bis- (p methoxybenzyl)-2-streptomycyl-tetrahydroimidazole sulfate separates and is collected by filtration.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in m1. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXVIH 1,3-bis-(2-thenyl) -2-strept0mycyZ-tetmhydroimidazole sulfate A solution of 2.5 grams of N,N'-bis-(2-thenyl)- ethylenediamine (diacetate melts about 84 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The

amount of ethanol is added to ensure complete solution. After heating at 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. After cooling, the crystalline product, 1,3 bis-(Z-thenyl)-2-streptomycyl-tetrahydroimidazole sulfate, is filtered ofi and dried.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXIX 1,3-bis-(Z-methyl-cyclohexylmethyl) -2-streptomycyltetrahydroimidazole sulfate A solution of 2.8 grams of N,N-bis-(2-methyl-cyclohexymethyD-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. Lyophilization of the reaction mixture leaves solid 1,3 bis (Z-methyl-cyclohexylmethyl)-2-strepto mycyl-tetrahydroimidazole sulfate.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXX 1,3-bis-(3 methyl cyclohexylmethyl)-2-streptomycyl tetrahydroimidazole sulfate A solution of 2.8 grams of N,N'-bis-(3-methyl-cyclohexylmethyl) -ethylenediamine dissolved in the minimum amount of methanol is added to 7 .3 g. streptomycin sulfate dissolved in 50 ml. Water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation in vacuo below 50 C. and replaced by water; upon cooling the product, 1,3 bis-(3-methyl-cyclohexyln1ethyl)-2-streptomycyl-tetrahydroimidazole sulfate, precipitates as a gum which gradually solidifies upon seeding.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble streptomycin.

14 EXAMPLE XXXI 1,3-bis (4 methyl-cyclohexylmethyl)-2-streptomycyltetrahydroimidazole sulfate A solution of 2.8 grams of N,N'-bis-(4-methyl-cyclohexylmethyD-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 4S50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The precipitate of 1,3-bis-(4-methyl-cyclohexylmethyl)-2- streptomycyl-tetrahydroimidazole sulfate which separates upon the addition of water is collected by filtration and dried.

A 1.5 gram sample of this imidazole is heated for three hours at C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXXn 1,3-bis-(4 methoxy-cyclohexylmethyl) 2-streptomycyltetrahydroz'midazole sulfate A solution of 3.1 grams of N,N-bis-(4-methoXy-cyclohexylmethyl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heat-- EXAMPLE XXXHI 1,3-bis-(3 methoxy-cyclohexylmethyl) -2-streptomycyltetrahydroimidazole sulfate A solution of 2.2 grams of N,N-bis-(3-methoxy-cyclohexylmethyl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45-50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-bis-(3-methoxy-cyclohexylmethyl)-2- streptomycyl-tetrahydroimidazole sulfate, is recovered as a solid 'oy lyophilization.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XX)C[V 1,3-bis 2(2 methyltheuyl)-2-strept0mycyl-tetrahydroimidazole sulfate A solution of 2.8 grams of N,N'-bis-2(2-methylthenyl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-bis-2(Z-methylthenyl) -2-streptomycyltetrahydroimidazole sulfate, precipitates upon cooling as a somewhat yellow oil which on standing becomes crystalline. The crystals are removed by filtration and dried in vacuo.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomy- EXAMPLE XXXV 1,3-bis-2 (quinolylethyl) -'2 -sireptontycyl-tetrahydrd i f imidazole sulfate A solution of 3.7 grams of N,N'-bis-2(quinolylethyl)- ethylenediamine dissolved inthe amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The

product, l,3;- bis-2(quinblylethyl)-2-streptomycyl-tetrahydroimida'zole sulfate, precipitates upon the addition of water and is filtered ed and dried;

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 2 5 ml. of 6 N HCl to regenerate,

the amine hydrochloride and soluble, active streptomy- 7 benzyl 3 alpha ethylbenzyl 2 streptomycyl zfetrahydroimidaz'ole sulfate A solution of 2.7 grams of N-benzyl;N'-alpha-ethylben zyl-ethylenediamine (dihydrochloride melts about 306 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added EXAMPLE. XXXVII.

1,3 bis (p methylbeuzyl) 2 streptomycyl tetrahydroimidazole sulfate A solution of 2.7 grams of N,N'-bis(p-methylbenzyl)- ethylenediamine (diacetate melts about 116 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. Water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. Upon cooling and the careful addition of water, the solid l,3-bis-(pmethylbenzyl)- 2-streptomycyl-tetrahydroin'iidazole sulfate separates and is collected by filtration.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XXXVIII hydroimidazole sulfate A solution of 2.7 grams of N,N-bis(m-methylbenzyl)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensurecomplete solution. heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. After cooling, the crystalline product, 1,3- bis (m-methylbenzyl) -2-streptomycyl-tettrahydroimidazole sulfate, is filtered .ofl and'dried.

A. 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

After '15 EXAMPLE XFQCIX 1,3 bis(0 methylbenzyl) -i2 stre'ptomycyl ttrahydroimidazole sulfate A solution of -2;7'grams of N,N-bis(o-methylbenzyl)- ethylenediamine dissolved'in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dis-. The minimum amount :of.

solved in 50 ml. water. ethanol is added 'to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for'three days. Lyophilization of the reaction mixture leaves solid 1,3 bis (o methylbenzyl) 2 streptomycyl tetrahydroimidazole sulfate. 1

A 1.5 gram sample of this imidazole is heated for'three hours at C. in 25ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XL 1,3 f bis (beta phenyl ethyb 2 strptomycyl tetra-' hydroimidazole sulfate A solution of 3.0 grams of N,N'-bis(beta-phenyletl1yl)-ethylenediamine '(diacetate melts about 114 C.)

dissolved in the minimum amount of methanol is added 7 to 7.3 g. streptomycin sulfate dissolved in 50 ml. water.

The minimum amount of ethanol is added to ensure complete solution. After heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation in vacuo below 50 C. and replaced by water;

upon cooling the product, 1,3-bis-(beta-phenylethyl) 2- streptomycyl-tetrahydroimidazole sulfate, precipitates as a gum which gradually solidifies upon seeding.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XLr 7 1,3 bis (lauryl) 2 streptomycyl tetraliydro imidazole sulfate A solution of 4.0 grams of N,N-bis-(lauryl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml.

Water. The minimum amount of ethanol is added to ensure hours at 100 C. in 25 ml. of 6 N HCl to regenerate'the amine hydrochloride and soluble, active streptomycin.

E A L L f 1,3 bis 3 methyl cyclohexyl) L 2 streptomycyl tetrahydro imiaa zole sulfate A solution of 2.5 grams of N,Nbis-(3-methylcyclohexyl)-ethylenediamine dissolved in the minimum amount V tetrahydroimidazole sulfate, is recovered as a nearly white solid by removing the solvents by distillation in vacuo.

A 1.5 gram sample of this irnidazole' is heated for three hours at 100 C. in 25 ml; of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

1? EXAMPLE XLIII 7 L3 bis (2 methylcyclohexyl) 2 streptomycyl tetrahydroimidazale sulfate A solution of 2.5 grams of N,N'-bis-(2-methylcyclohexyl) ethylenediamine dissolved in the minimum amount of methanol is added to 7 .3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 :C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3 bis (2 methylcyclohexyl) 2 streptornycyl-tetrahydroimidazole sulfate, is recovered as a solid by lyophilization.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XLIV 1 ,3-bis-(4-methylcycl0hexy) -2-strept0mycyltetrahydroimidazole sulfate A solution of 2.5 grams of N,N-bis-(4-methylcyclohexy1)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45-50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3 bis (4 methylcyclohexyl)-2-streptomycyltetrahydroimidazole sulfate, precipitates upon cooling as a somewhat yellow oil which on standing become crystalline. The crystals are removed by filtration and dried in vacuo.

EMMPLE XLV 1 ,3-bis-(3-tnitr0plzenyl -2-sZrept0mycyl-tetrahydroimidazole sulfate A solution of 3.3 grams of N,N'bis-(3-nitrophenyl)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50. ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3 bis (3-nitrophenyl)-2-streptomycyl-tetrahydroimidazole sulfate, precipitates upon the addition of water and is filtered off and dried.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XLVI 1,3-bis-2-(6-methylpyridyl) -2-streptomycyl-tetrahydroimidazole sulfate A solutions of 2.4 grams of N,N'-bis-2-(6-methylpyridyl) -ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sul fate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The solid product, 1,3-bis-2-(6-methylpyridyl)- Z-streptomycyl-tetrahydroimidazole sulfate, precipitates upon seeding and the addition of water and is collected by filtration and dried.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XLVII 1,3 bis-2-(5-methylpyridyl)-2-strept0mycyLtetrahydroimidazale sulfate A solution of 2.4 grams of N,N-bis-2-(5-methylpyridyl)-e'thylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol EXAMPLE XLVIII Y ,3-bis-2 (4- methylpyridy l -2 -streptomycyl-tetrahydroimidazole sulfate A solution of 2.4 grams of N,N-bis-2-(4-methylpyridyl)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. After cooling, the crystalline product, 1,3-bis-2-(4-methylpyridyl) -2- streptomycyl-tetrahydroimidazole sulfate, is filtered off and dried.

A 1.5 gram sample of this imidazole is heated for three hours at C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE XLIX 1,3 bis-2-(3 methylpyrz'dyl) -2-streptomycyl tetrahyaroimidazole sulfate A solution of 2.4 grams of N,N-bis-2-(3-methyl- .pyridy1)-ethylenediamine dissolved in the minimum EXAMPLE L 1 ,3 -bis-2-thiaz0lyl-2 -strept0mycyl-tetrahydroimia azole sulfate A solution of 2.3 grams of N,N'-bis-2-thiazolyl-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 grams streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation in vacuo below 50 C. and replaced by water; upon cooling the product, l-3-bis-2-thiazolyl-2-streptomycyl-tetrahydroimidazole sulfate, precipitates as a guru which gradually solidifies upon seeding.

A 1.5 gram sample of this imidazole is heated for three hours at l00 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptd mycin.

EXAMPLE LI 1 ,3 -bis-2 (5 -methyl furyl -2-streptomycyl-tetrahydroimidazole sulfate amass EXAMPLE LII 1,3-bis- (p-hydroxybenzyl) -2-strept0mycyl-tetrahydroimidaz ole sulfate A solution of 2.7 grams of N,N'-bis-(p-hydroxybenzyl)- ethylenediamine (dihydrochloride meltsjat about 242 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. Water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-bis-(phydroxybenzyl)-2-streptomycyl-tetrahydroimidazole sulfate, is recovered as a nearly White solid by removing the solvents by distillation invacuo.

A 1.5 gram sample of this .imidazole is heated. for three hours at 100 C. in 25 ml. of 6 N'HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LEI 1,3 bis (cyclopentyl) 2 streptomycyl tetrahydroimidazole sulfate w A solution of 2.0 grams of .N,N-bis-(cyclopentyl)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin'sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3 bis (cyclopentyl) Z-streptomycyl-tetrahydroimidazole sulfate, is recovered as a solid by lyophilization.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LIV 1,3 bis (undecyl) 2 streptomycl tetrahydro imz'dazole sulfate A solution of 3.7 grams of N,N-bis(undecyl)-ethylenediaminedissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 -50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3- bis-(undecyl)-2-streptomycl-tetrahydroimidazole sulfate, precipitates upon cooling as a somewhat yellow oil which on standing becomes crystalline. The crystals are removed by filtration and dried in vacuo.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LV 1,3 bis (4 methoxycyclohexyl) 2 streptomycl tetrahydroimidazole sulfate A 1.5 gram sample of this irnidazole is 'heated f lf "2o 7 A three hours at 100 C. in ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LVI' 1,3 bis (vanillyl) 2 streptomycl tetruhydroimidazole sulfate A solution of 3.3 grams of N,N-bis-(vanillyl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml;

water. The minimum amount of ethanol is added toensure complete solution. After heating at 45 -50 C. for 4 ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The solid product, 1,3 bis (vanill yl) 2 streptomycyl tetrahydroimidazole sulfate, precipitates upon seeding and the'addition' of water and is collected by filtration and drieds V A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LVII 1 cyclohexyl 3 ethyl 2 streptomycyl te trahydroimz'dazole sulfate hours at C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LVIII 1 benzyl 3 vunillyl 2 streptomycyl tetrahydroimiduzole sulfate A solution of 2.9 grams of N-benzyl-N-vanillyl-ethyl enediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water.. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. After cooling, the crystalline product, 1 benzyl 3 vanillyl 2 streptomycyl-tetrahydroimidazole sulfate, is filtered off and dried.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LIX 7 1,3 din butyl 2 streptomycyl tetrahydroimidazole sulfate hours at 100 C. in 25 ml. of 6 N HCl to regenerate the.

amine hydrochloride and soluble, active streptomycin.

EXAMPLE LX 1,3 di iso butyl 2 streptomycyl tetrahydroimidazole sulfate A solution of 1.7 grams of N,N'-di-iso-butyl-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. Water. The minimum amount of ethanol is added to ensure complete solution. After heatingat45" -.50 "C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The alcohol is removed by distillation in vacuo below 50 C. and replaced by water; upon cooling the product, 1,3-di-iso-butyl-2-streptomycyl-tetrahydroirnidazole sulfate, precipitates as a gum which gradually solidifies upon seeding.

A 1.5 gram sample of this'imi'da'zole is heated for three hours at 100 C. in 25 ml. of 6N 'HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXI 1,3 di sec. butyl-Z-streptomyeyl tetrahydrdirfiidazole sulfate A solution'of 1.7 grams of 'N,N-di sec.-butyl-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at ,room temperature for three days. The precipitate of 1,3 di sec.-butyl 2 streptomycylatetrahydroimidazole sulfate which separates upon the addition of water is collected by filtration and dried.

A 1.5 gram sample of this imidazoleis heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXII 1,3-bis-(1-naphthyl) -2-strept0mycyl tetrahydroimidazole sulfate A solution of 3.1 grams of N,N "-bis-(.l-naphthyl-)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. Water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45-50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The precipitate of 1,3-bis-(l-naphthyl)-2-streptomyoyltetrahydroimidazole sulfate is recovered as a nearly white solid by removing the solvents by distillation in vacuo.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LX111 1,3-bis-(2-naphthyl)-Z-strept0mycyl tetrahytlro'imidazole sulfate A solution of 3.1 grams of N,N'-bis-(2-naphthyl)- ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum of ethanol is added.

EXAMPLE LXIV 1,3-bis-(2-pyridyl) 2 streptom-ycyl-tetrahydroimidazole sulfate A solution of 2.1 grams of N,N'-bis-(2-pyridyl)-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 1,3-bis-(2-pyridyl) 2 streptomycyl-tetrahydroimidazole sulfate, is recovered as a solid by lyophilization.

A 1.5 g. sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EMMPLE LXV 1,3-bisbenzhydryl) -2-streptomycyl tetrahydroim'idazole sulfate A solution of 3.9 grams of N,N'-bis-(benzhydryl)- ethylenediamine dissolved in the minimum amount of methanolis added to 7.3 g. streptomycin .sulfatedissolved in "50 m1. water. The amount "of ethanol .is added to ensure complete solution. After heating at 4'550 'C. for teh 'minutes, the "reaction mixture is allowed to stand fat room temperature'for three days. The product, l,3-bis(benihydryl)e2-streptomycyl tetrahydroimidazole sulfate, precipitates upon cooling as a somewhat yellow oil which on standing becomes crystal line. The crystals are removed by filtration and dried in vacuo. I V

A 1.5 gram sample of this 'imida'zole is heated for three hours at C. in 25 ml. of 6 N 'HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXVI 1 ,3-bis- (3 ,4 -d imethoxy benzyl) -2- s trept0mycyl-tetrahydro- .imidazole sulfate A solution of 3.6 grams of N,N-bis(3,4-dimethoxybenzyD-ethylenediamine dissolved in theminimum amount of methanol is added to 7.3 g.

EXAMPLE 'LXV II 1 benzyl-3-p-meth'0xybenzyl-Z-streptcimycfl tetrahydrdimidazole sulfate i A solution of 2.7 grams of .N-benzyl-Np-methoxybenzylethylenediarnine dissolved in the minimum amount of methanol is added to 7.3g. streptomycin sulfate dissolved in 50 ml. 'water. The minimum amount ofethanol is added to ensure complete solution. After heating at 45 50 C. .for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, l-benzyl-3-p-methoxybenzyl-2-streptomycyl tetrahydroimidazole sulfate, precipitates upon the addition of water and is filtered off and dried.

A 1.5 g. sample 'of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl-to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXVlll -1,3-bis- (amyl) -2-strep t0mycyl-tetrahydroimidazole sulfate A solution of 2.0 grams of N,N'-bis(amyl')-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The solid product, 1,3 bis (amyl)-2-streptomycyl-tetrahydroimidazole sulfate, precipitates upon seeding and the addition of water and is collected by filtration and dried.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXlX I-ethyl 3 phenethyl-Z-streptomycyltetrahydr0imidaz0le sulfate A solution of 1.9 grams of Neethyl-N-phenethyl-ethylenediarnine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. Upon cooling and the careful addition of Water, the solid 1-ethyl-3- phenethyl-Z-streptomycyl-tetrahydroimidazole sulfate separates and is collected by filtration.

A 1.5 gram sample of this imida zole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXX 1,3 bis (orthochlorobenzyl) -2-streptomycyl-tetrahydroimidazole sulfate I A solution of 3.1 grams of N,N'-bis-'(orthochlorobenzyl)-ethylenediamine (dipenicillin G saltmelts about agar; 11s

.tomycyl-tetrahydroimidazole sulfate; is filtered off and dried.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE 1,3 bis (orthohydroxybenzyl) L 2 streptomycyl tetrahydroimidazole sulfate A solution of'2.7 grams of N,N'-bis-(orthohydroxybenzyD-ethylenediamine (dipenicillin G salt melts about 115 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml'. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. 'for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. 'Lyophilization of the reaction mixture leaves solid 1,3?bis-(orthohydroxybenzyl)-2-streptomycyl-tetrahydroimidazole sulfate.

' A 1.5 gram sample of this imidazole is heated for three "hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXXIL 1 benzyl 3 (2 hydroxylbenzyl) 2 streptomycyltetrahydroimidazole sulfate moved by distillation in vacuo below 50 C. and replaced by water; upon cooling the product, l-benzyl-3-(2-hydroxylbenzyl) 2 streptomycyl tetrahydroimidazole sulfate, precipitates as a gum which gradually solidifies upon seeding.

A 1.5 gram' sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE 'LXXIII 1 -benzyl-3- (3-ethoxy-4-hydroxybenzyl -2-strept0mycyltetrahydroimidazole sulfate A solution of 3.0 grams of N-benzyl-N-(3-ethoxy-4- hydroxybenzyl) -ethylenediamine (dipenicillin G salt melts about 120 C.) dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C..for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The precipitate of 1-benzyl-3-(3-ethoxy-4-hydroxybenzyl)-2- streptomycyl-tetrahydroimidazole sulfate which separates upon the addition of water is collected by filtration and dried.

A 1.5 gram sample of this imidaZole is heated for three hours at 100 C. in.25 ml. of 6 N I-ICl to regenerate the amine hydrochloride and soluble, active streptomycin.

I, 24 EXAMPLE LXXIV 4-methyl-1,3-di-n-heptyl-2-streptomycyl-tetrahydro- V imidazole sulfate A solution of 2.7 grams of 1-methyl-N,N'-di-n*heptylethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, imidazole sulfate, is recovered as a nearly white solid by removing the solvents by distillation in vacuo. f,

A 1.5 gram sample of this imidazole is heated for three hours at C. in 25 ml. of 6 N HCI to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXXV 4,5-dimetlz'yD-1,3-dibenZyI-Zstreptomycyl-tetrahydro imidazole sulfate A solution of 2.6 grams of LZ-dimethyl-N,Nedibenzylethylenediamine. dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After'heating at 45- 50 C..for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The prod EXAMPLE LXXVI 4-methyl-l,3-dibenzyl-2-strept0mycyl-tetrahya'roimidazole sulfate A solution of 2.5 grams of 1-methyl-N,N -dibenzylethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 '50' C. for ten minutes, the reaction mixture is allowed to stand at room temperature forthree days. The product, 4 methyl 1,3 dibenzyl 2 streptomycyl tetrahydroimidazole sulfate, precipitates upon cooling as a somewhat yellow oil which on standing becomes crystalline. The crystals are removed by filtration and dried in vacuo.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

' EXAMPLE LXXVII 4,5-dimethyl-1,3-di-n-heptyl-2-streptomycyl-tetrahydroimidazole sulfate A solution of 2.8 grams of l,2-dimethyl-N-N'-di-nheptyl-ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45 50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. The product, 4,5 dimethyl 1,3 di n heptyl 2 streptojmycyltetrahydroimidazole sulfate, precipitates upon the addition of water and is filtered off and drie A 1.5 gram sample of this imidazole is heated for three The minimum amount of ethanol is 4-methyl-1,3-di-n-heptyl-2-streptomycyl-tetrahydrotemperature for three days. 'rnethyl -l,3 dicyclohexylmethyl 2 -'s treptomycyltetradissolved in the amount of methanol is added to g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added toensurecompleteisolution. Aftersheating at "4'5 '50 C. for'ten minutes, the reaction :mixture 'is allowed to stand at 'room The solid product, 4;5-dihydroimidazole sulfate, precipitates upon seeding'and'the additionof'water and is collected "by-filtration and dried.

A -l.-5 gram sample of this imidazole -is heated for three hours'at 100C. in'25 ml. of 6-N 'HCl to regenerate the aminejhydrochloride-andsoluble, active streptomycin.

EXAMPLE LXXIX 4 methyl l ,3 di cyclohexylmethyl 2 streptomycyltetrahydroimidazo'le sulfate 4- methyl l ,3 di --cyclohexylmethyl-- 2 streptomyeyltetrahydroimidazole sulfate separates and is collected by fi ra 'A 1.5 gram sample of this imid azole'is'heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride .and soluble, active streptomycin.

EXAMPLE LXXX 4,5 dimethyl 1,3 di-cyclohexyl 2 streptomycyltetrahydroimidazole sulfate A solution .of 2.4 grams of 1,2-dimethyl-N,N'-di-cyclohexyl ethylenediamine dissolved in the minimum amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 45-50 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. After cooling, the crystalline product, 4,5dimethyl1,3 di-cyclo'hexyl-2-streptomycyl-tetrahydroimidazole sulfate, is filtered off and dried.

A 1.5 gram sample of this imidazole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXiCXl 4 methyl 1,3 di cyclohexyl 2 streptomycyl-tetrahydroimidazole sulfate A solution of 2.3 grams of 1-methyl-N,N'-di-cyclohexyl-ethylenediamine dissolved in the amount of methanol is added to 7.3 g. streptomycin sulfate dissolved in 50 ml. water. The minimum amount of ethanol is added to ensure complete solution. After heating at 4550 C. for ten minutes, the reaction mixture is allowed to stand at room temperature for three days. Lyophilization of the reaction mixture leaves solid 4- methyl 1,3 di cyclohexyl 2 streptomycyl tetrahydroimidazole sulfate.

A 1.5 gram sample of this imid azole is heated for three hours at 100 C. in 25 ml. of 6 N HCl to regenerate the amine hydrochloride and soluble, active streptomycin.

EXAMPLE LXXXH 1,3 dibenzyl 2 streptomycyl tetrahydroimidazole sulfate Eight grams of solid streptomycin sulfate (po tency475 units/mgm. by B. subtilis assay; partially purified by adsorption on and elution from ion-exchange resin columns) was dissolved in 50 ml. water and mixed with a solution of 10 mls. N,N- dibenzyl-ethylenediamine in 70 mls. methanolto give a clear solution which was mycyl-tetrahydroimidazole sulfate (7.64 grams) precipitated and was collected ".by filtration, dried over P205 and found to assay '600 units/mgm. The filtrate (78 mls.) assayedZSSO-units/ml.

7.5 .grams :of this 1,3-dibenzyl-2-streptomycyl-tetrahydroimidazole sulfate were placed in water and the pH .was adjustedtopH 2 with sulfuricacid. N,Ndibenzylethylenediamine sulfate precipitated andwascollected'by filtration (1.'04.g.). Addition offiv'e volumes ot-methanolto the filtrateprecipitated rsolid streptomycin sulfate (4.08 .g.) assaying 625 u/mgm.

' LXXXIII .Fifty mls. of streptomycin liquor gassaying 182,000 units/ml. (B. subtilis assay) and obtained by elution of broth adsorbed on an ion-exchange resin column was sayed .2480 units/ ml.

Four grams of this l,3-dibenzylQ-streptomycyl tetrahydroim'idazole sulfate was ,placed in water and the .pH

was adjusted to pH 2.0 withsulfuric acid. .Afterstand- 'ing, the volume was reduced by one-half by distillation in vacuo and the N,N'-dibenzyl-ethylenediamine sulfate which precipitated (0.92 g.) was removed by filtration. The addition of five volumes methanol precipitated 2.53 g. streptomycin sulfate assaying 560 units/mgm; the filtrate (925 ml.) assayed 122 units/ml.

1,3-dibenzyl-2-streptomycyl-tetrahydroimidazole sulfate (2%) in 4% aqueous acacia suspension has LDso (minimum lethal dose in 50% of animals) of about 3421-21 mgm./kg. by intraperitoneal injection in mice. Streptomycin sulfate, 1,3-dibenzyl-2-streptomycyl-tetrahydroimidazole sulfate and 1,3-di(beta-phenylethyl)-2 streptomycyl-tetrahydroimidazole sulfate by in vitro test all have minimum inhibitory concentrations of 0.0002 mgm./ml. against the streptomycin-sensitive strain H37RU of Mycobacterium tuberculosis and all fail to inhibit streptomycin-resistant strain H37RUR.

The CD50 (Curative Dose-50) is the minimum intraperitioneal dose of the drug which will cure 50 percent of a group of mice injected intraperitioneally with to 1000 LD50 doses of Diplococcus pneumoniae, each LDso dose being suificient if given alone to kill 50 percent of a group of mice. One-half the dose of test drug is given twenty-four hours before the simultaneous injection of the other half of the test drug and the challenge (infection). The animals are observed for four days and deaths for each group expressed as the percentage of the total animals per group. The percentage death is transformed to probit values and these plotted against the log of the dose in mgms. per kg. of mouse weight. The point of intersection of the probit 5 line and the best line constructed through the experimental points describes the concentration of drug which should protect half the animals under the conditions of the experiment. The antilog of this term is called the CD50 value. Using aqueous suspensions, including sodium carboxymethylcellulose and having pH about 7, of the two new drugs, the following values for CD50 were found as above: 6.8 mgm./kg. for streptomycin sulfate; 45 mgm./kg. for 1,3- dibenzyl-2-streptomycyltetrahydroimidazole sulfate; and 60 mgm./kg. for 1,3-di(betaphenethyl)-2-streptomycyltetrahydroimidazole sulfate.

I claim:

1. The process of purifying streptomycin comprising reacting in alkaline solution streptomycin with a diamine selected from the group consisting of compounds having the formula R3 v R1NH(EHCHNHR2 wherein R1 and R2 represent radicals selected from the group consisting of alkyl, cyclopentyl, cyclopentyl-lower alkyl, cyclohexyl, cycloheXyl-lower alkyl, lower alkylcyclohexyl-lower alkyl, lower alkoxy-cycloheXyl-lower alkyl, 'lower alkyl-cyclohexyl, lower alkoxy-cyclohexyl,

'dehydroabietyl, pyridyl, lower; alkyl-pyridyl, thiophenelower alkyl, lower alkyl-thiophene-lower alkyl, furanlower alkyl, lower akyl-furan-lower alkyl, quinolyllower alkyl, naphthyl, benzhydryl, piperonyl, thiazolyl,

phenyl, lower alkyl-phenyl-lower alkyl, phenyl-lower 7 alkyl, halophenyl-lower alkyl, nitrophenyl-lower alkyl, hydroxyphenyl-lower alkyl, HzN-phenyl-lower alkyl, lower 'alkoxy-phenyl-lower alkyl, alkoXy-hydroxy-phenyllower alkyl, and 'di-lower alkyl-monohydroxy-phenyllower alkyl'; R3 and R4 'representmembers selected from the group consisting. of hydrogen and methyl, isolating 'the product so formed and regenerating 'purifie'dstreptomycin by hydrolyzing this product in an acid medium.

" 2. The. process of purifying streptomycin comprising reacting in alkaline solution streptomycin with N,N-dibenzylethylencdiamine, isolating the product so formed and regenerating purified streptomycin by hydrolyzing this product in an acid medium.

streptomycin comprising reacting in alkaline solution an acid addition. salt of streptomycin with N,N'-dibenzylethylenediamine, isolating the product so formed and regenerating said purified acid addition-salt of streptomycin by hydrolyzing this product in an acid medium a which is acidified with the same acid which forms said acid addition salt of streptomycin.

4. The process of purifying streptomycin sulfatecomprising reacting, in alkaline solution, streptomycin sul- 3. The process of purifying an acid addition saltof fate with N,N-dibenzylethylenediamine, isolating the a product so formed and regenerating purified streptomycin sulfate by hydrolyzing this product in an acid medium which is acidified with sulfuric acid. 7

References Cited in the file of h Pa t v V.

UNITED STATES PATENTS OTHER REFERENCES Alphen: Rec. Trav. Chirn., volume 55 (1936), pages 

1. THE PROCESS OF PURIFYING STREPTOMYCIN COMPRISING REACTING IN ALKALINE SOLUTION STREPTOMYCIN WITH A DIAMINE SELECTED FROM THE GROUP CONSISTING OF COMPOUNDS HAVING THE FORMULA 